Vascular endothelial growth factor/semaphorin-3A ratio and SEMA3A expression in cutaneous malignant melanoma.

Breslow thickness and Clark level are still important factors for cutaneous melanoma, but do not provide a precise prognosis in all cases. It is necessary to find new factors capable of a more accurate prediction of the tumor course. Angiogenesis is essential for tumor development and progression and is regulated by vascular endothelial growth factor A (VEGF-A) and semaphorins (SEMA), in particular, SEMA3A inhibits angiogenesis by affecting VEGF signaling. However, the prognostic role of angiogenetic factors remains unclear. To date, no information is available on SEMA3A in human melanoma. Microvessel density, immunohistochemical and mRNA VEGF and SEMA3A expression level in 60 thin (Breslow thickness ≤ 1.0 mm), 60 intermediate (1.1-4.0 mm) and 50 thick (>4.0 mm) primary human cutaneous melanomas were investigated and related to clinical/pathological parameters and disease-specific survival. No positive association between Breslow thickness, Clark level, metastasis presence and survival was identified; Clark level was poorly related to survival. VEGF and microvessel density were significantly higher in intermediate and thick melanomas and related to Breslow thickness and Clark level but not to metastasis status and survival. On the contrary, SEMA3A was significantly reduced in intermediate and thick melanomas and associated to metastasis and poor survival. VEGF/SEMA3A ratio was higher in the worst prognosis, resulting the most closely related factor with metastasis and survival. SEMA3A expression and VEGF/SEMA3A ratio turned out to be valuable prognostic biomarkers in patients affected by cutaneous melanoma, in particular with Breslow thickness >1 mm. SEMA3A might serve as a candidate tumor suppressor in cutaneous melanoma therapy.

A bone-targeting drug-delivery system based on Semaphorin 3A gene therapy ameliorates bone loss in osteoporotic ovariectomized mice.

Osteoporosis is a serious health problem worldwide. Semaphorins (Sema) have been described as key molecules involved in the cross-talk between bone cells (osteoblasts/osteoclasts). In this study, we investigated whether plasmid containing Sema3a could ameliorate bone loss in an ovariectomized (OVX) mouse model via (AspSerSer)6, a selectively bone-targeting moiety. Plasmid pcDNA3.1(+)-Sema3a-GFP was fabricated and transfected cells with the plasmid demonstrated statistically higher levels of Sema3A in vitro (p < 0.001). Mice were ovariectomized and injected twice weekly with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP for four weeks. The aim of the study was twofold: firstly to design an effective bone-targeting drug-delivery system (AspSerSer)6. Secondly, the effects of Sem3A gene therapy on bone loss was investigated. Here, the targeting selectivity of pcDNA3.1(+)-Sema3a-GFP via (AspSerSer)6 to the trabecular bone surface was firstly verified by histological observation of frozen sections and immunofluorescence staining. Then, bone microstructure analysis by Micro-CT indicated significantly less bone loss in mice treated with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP compared to the control group (p < 0.05). Furthermore,H&E staining and Safranin O staining of the decalcified sections demonstrated statistically significantly higher bone area/total area in the mice that were injected with (AspSerSer)6-(STR-R8)+pcDNA3.1(+)-Sema3a-GFP (p < 0.001, p < 0.01,respectively). TRAP staining and immunohistochemistry staining of COL I demonstrated lower numbers of osteoclasts and significantly increased numbers of osteoblasts in the bone-targeting moiety delivering pcDNA3.1(+)-Sema3a-GFP group, when compared to the control group (p < 0.01, p < 0.001,respectively). Together, our findings have identified that, (AspSerSer)6, a bone-targeting drug-delivery system based on semaphorin3A gene therapy, ameliorated bone loss in osteoporotic ovariectomized mice, by suppressing osteoclastic bone resorption and simultaneously increasing osteoblastic bone formation. Gene therapy by local site-specific Sema3A overexpression might be a potential new strategy for treating osteoporosis and bone defects.

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS.

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non-cell-autonomous mechanism of motor neuron degeneration in ALS.SIGNIFICANCE STATEMENT Despite some progress, currently no effective treatment is available for amyotrophic lateral sclerosis (ALS). We suggest a novel regulatory role for miR126-5p in ALS and demonstrate, for the first time, a mechanism by which alterations in miR126-5p contribute to axon degeneration and NMJ disruption observed in ALS. We show that miR126-5p is altered in ALS models and that it can modulate Sema3 and NRP protein expression. Furthermore, NRP1 elevations in motor neurons and muscle secretion of Sema3A contribute to axon degeneration and NMJ disruption in ALS. Finally, overexpressing miR126-5p is sufficient to transiently rescue NMJ disruption and axon degeneration both in vitro and in vivo.

Genetic targeting of Purkinje fibres by Sema3a-CreERT2.

The maintenance of the heart rhythm and the conduction of excitatory signals require changing excitatory signals via electrical activity and coordination by communication between working and conductive cardiomyocytes. Understanding how the ventricular conduction system is established provides novel insights into the pathophysiological progress of cardiac arrhythmias. However, the major hurdle in this field is the lack of a specific genetic tool that targets the Purkinje fibres of the ventricular conduction system and no other types of cardiomyocytes or coronary vessels. Here, we generated a Sema3a-CreERT2 knock-in mouse line to test its specificity for genetically labelled Purkinje fibres. We found that Sema3a was expressed in the subendocardial layer of the trabecular myocardium in the embryonic heart and was restricted to the Purkinje fibres in the adult heart. A fate mapping study based on the Sema3a-CreERT2 line revealed that the Sema3a+ cardiomyocytes were restricted to the fate of Purkinje fibres in the perinatal but not the embryonic stage. Collectively, our study provides a new genetic tool, i.e., Sema3a-CreERT2, for studying the molecular mechanisms that regulate the function of Purkinje fibres.

Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma.

Semaphorin3A (SEMA3A), an axon guidance molecule in the nervous system, plays an inhibitory role in oncogenesis. Here, we investigated the expression pattern and biological roles of SEMA3A in head and neck squamous cell carcinoma (HNSCC) by gain-of-function assays using adenovirus transfection and recombinant human SEMA3A protein. In addition, we explored the therapeutic efficacy of SEMA3A against HNSCC in vivo. We found that lower expression of SEMA3A correlated with shorter overall survival and had independent prognostic importance in patients with HNSCC. Both genetic and recombinant SEMA3A protein inhibited cell proliferation and colony formation and induced apoptosis, accompanied by decreased cyclin E, cyclin D, CDK2, CDK4 and CDK6 and increased P21, P27, activated caspase-5 and caspase-7. Moreover, over-expression of SEMA3A suppressed migration, invasion and epithelial-to-mesenchymal transition due in part to the inhibition of NF-κB and SNAI2 in HNSCC cell lines. Furthermore, intratumoral SEMA3A delivery significantly stagnated tumor growth in a xenograft model. Taken together, our results indicate that SEMA3A serves as a tumor suppressor during HNSCC tumorigenesis and a new target for the treatment of HNSCC.

Semaphorin-3a, neuropilin-1 and plexin-A1 in prosthetic-particle induced bone loss.

Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. STATEMENT OF SIGNIFICANCE: Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.

Synapsin III acts downstream of semaphorin 3A/CDK5 signaling to regulate radial migration and orientation of pyramidal neurons in vivo.

Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation.

Withania somnifera Improves Ischemic Stroke Outcomes by Attenuating PARP1-AIF-Mediated Caspase-Independent Apoptosis.

Withania somnifera (WS), popularly known as "Ashwagandha" has been used for centuries as a nerve tonic. Its protective effect has been elucidated in many neurodegenerative pathologies, although there is a paucity of data regarding its effects in ischemic stroke. We examined the neuroprotective properties of an aqueous extract of WS in both pre- and poststroke treatment regimens in a mouse model of permanent distal middle cerebral artery occlusion (pMCAO). WS (200 mg/kg) improved functional recovery and significantly reduced the infarct volume in mice, when compared to those treated with vehicle, in both paradigms. We investigated the protective mechanism/s induced by WS using brain cortices by testing its ability to modulate the expression of key proteins in the ischemic-apoptotic cascade. The Western blots and immunofluorescence analyses of mice cortices revealed that WS upregulated the expression of hemeoxygenase 1 (HO1) and attenuated the expression of the proapoptotic protein poly (ADP-ribose) polymerase-1 (PARP1) via the PARP1-AIF pathway, thus preventing the nuclear translocation of apoptosis-inducing factor (AIF), and subsequent apoptosis. Semaphorin-3A (Sema3A) expression was reduced in WS-treated group, whereas Wnt, pGSK3ß, and pCRMP2 expression levels were virtually unaltered. These results indicate the interplay of antioxidant-antiapoptic pathways and the possible involvement of angiogenesis in the protective mechanism of WS while emphasizing the noninvolvement of one of the prime pathways of neurogenesis. Our results suggest that WS could be a potential prophylactic as well as a therapeutic agent aiding stroke repair, and that part of its mechanism could be attributed to its antiapoptotic and antioxidant properties.

Decreased expression of SEMA3A is associated with poor prognosis in gastric carcinoma.

BACKGROUND/PURPOSE: SEMA3A (semaphorin-3A), is a secreted protein that belongs to the semaphorin family and can function as both a chemoattractive agent or a chemorepulsive agent. SEMA3A has been shown to be a tumor suppressor in various cancers. This study investigated the expression of SEMA3A in gastric cancer and its prognostic value for gastric cancer patients. METHODS: We examined the expression of SEMA3A in paired cancerous and matched adjacent noncancerous gastric mucosa tissues by real-time quantitative RT-PCR (qRT-PCR) and western blotting. In vitro, we evaluate the effects of SEMA3A on gastric cancer cell proliferation and migration by MTT, transwell and wound-healing assays. Furthermore, we analyzed SEMA3A expression in 128 patients who underwent resection procedures using immunohistochemistry. The relationships between the SEMA3A expression levels, the clinicopathological factors, and patient survival were investigated. RESULTS: Our results revealed decreased SEMA3A mRNA (P = 0.0037) and protein (P = 0.033) expression in tumor tissue samples compared with matched adjacent non-tumorous tissue samples. Overexpression of SEMA3A inhibits gastric cancer cell proliferation and migration in vitro. Immunohistochemical staining data showed that SEMA3A expression was significantly decreased in 54.68% of gastric cancer cases. In addition, the chi-square test revealed that low SEMA3A expression was significantly correlated with poor differentiation (P = 0.015), Vascular invasion (P = 0.001), depth of invasion (P < 0.001), lymph node metastasis (P = 0.029), distant metastasis (P = 0.002) and advanced TNM stage (P = 0.003). SEMA3A expression was positively correlated with clinical TNM stage, that suggested the more advanced clinical TNM stage corresponding to the lower expression level of SEMA3A (rs = -0.322, P < 0.001) by Spearman rank correlation analysis. Kaplan-Meier survival analysis demonstrated that low expression of SEMA3A was significantly correlated with a poor prognosis for gastric cancer patients (P < 0.001). The multivariate analysis revealed that SEMA3A expression was an independent prognostic factor of the overall survival rate of patients with gastric cancer. CONCLUSION: SEMA3A expression decreased significantly as gastric cancer progressed and metastasized, suggesting that SEMA3A might serve as a candidate tumor suppressor and a potential prognostic biomarker in gastric carcinogenesis.

Effect of endostar combined with cisplatin on expression of VEGF and Sema3A of Lewis lung cancer rats.

OBJECTIVE: To evaluate the therapeutic effect of endostar (ED) combined with cisplatin(DDP) on model of C57BL/6 rats, and to further investigate the inhibiting mechanism of endostar from tumor angiogenesis. METHODS: Lewis lung cancer cells were inoculated in C57BL/6 mouse, then the mouse were randomized into control group (group A), ED (group B), DDP (group C) and ED/DDP (group D). They were treated according to the plan. And the expressions of VEGF and Sema3A were evaluated by immunhistochemisty. RESULTS: The weight of tumor increased in group A and B. It was decreased in group C and D. The tumor volume was increased in all the 4 groups. The VEGF expression of group D was obviously lower than the other group 3, but the Sema3A expressed of group D was significantly strengthener than the other group 3. The VEGF expression of group B and group D were obviously low especially in the 4th-8th days. Pearson correlated analysis showed that the expression VEGF and Sema3A were negatively correlated (r=-0.72, P<0.05). CONCLUSIONS: ED combined with DDP could control the tumor growth effectively, and avoid weight loss. ED could reduce VEGF expression, and enhance Sema3A expression. Tumor vessel presents transient normalization. It is easy for DDP perfusion, and to kill tumor cells.

Expression of semaphorin 3A and neuropilin 1 with clinicopathological features and survival in human tongue cancer.

OBJECTIVE: To investigate the association between semaphorin 3A (SEMA 3A) and its receptor neuropilin 1 (NRP1) and the clinicopathologic characteristics of patients with tongue cancer. STUDY DESIGN: Forty-three tongue squamous cell carcinoma specimens were included. Immunohistochemical staining of SEMA3A and NRP1 was performed on 15 normal tongue epithelium specimens and the 43 tumour specimens. Immunoreactivity was evaluated based on the staining intensity and distribution score. Statistical analyses were performed using Chi-squared and Spearman tests and Kaplan-Meier analysis. RESULTS: SEMA3A was significantly down-regulated in tongue cancer compared with normal tongue (P=0.025), while NRP1 was over-expressed in tumours (P<0.001). SEMA3A expression inversely correlated with nodal metastasis (P=0.017). NRP1 expression did not correlate with any clinicopathological characteristics. Higher SEMA3A expression strongly predicted longer survival (P=0.005). Scores for the NRP1/SEMA3A ratio of ≥1 predicted shorter survival (P=0.045). CONCLUSIONS: Aberrant expression of SEMA3A and its receptor NRP1 might be involved in the development of tongue cancer and might be useful prognostic markers in this tumour type.

Semaphorin 3A overcomes cancer hypoxia and metastatic dissemination induced by antiangiogenic treatment in mice.

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

Semaphorin 3A expression in the colon of Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by an absence of intrinsic ganglion cells in the nerve plexuses of the lower colon. The Semaphorin 3A (SEMA3A) gene is involved in the migration of enteric neural precursors (ENPs). To analyze the function of SEMA3A in HSCR, the SEMA3A expression in different colon segments in HSCR was examined. METHODS: The expression levels of SEMA3A in both ganglionic and aganglionic colon tissues of 32 patients with HSCR and in colon tissue of 5 newborn unaffected individuals were examined by real-time RT-PCR, Western-blot, and immunohistology. RESULTS: Comparison of SEMA3A expression levels between ganglionic and aganglionic tissues in HSCR revealed upregulation of SEMA3A expression in 43.75% (14/32) of the aganglionic colons. SEMA3A was expressed in the ganglion cells of the myenteric plexus, submucosa, as well as in the longitudinal and circular muscle layer of the normal colon of both unaffected newborns and patients with HSCR. In the aganglionic segment of patients with HSCR, SEMA3A was highly expressed in the circular muscle layer and was also detected in the submucosa and in the longitudinal muscles layer. The fluorescence intensity of SEMA3A in the circular muscle layer in the aganglionic segment was much higher than that in ganglionic segment (p < .001). CONCLUSION: SEMA3A expression was upregulated in the aganglionic smooth muscle layer of the colon in some patients with HSCR and our data suggest that increased SEMA3A expression may be a risk factor for HSCR pathology in a subset of patients.

Syntaxin 1B suppresses macropinocytosis and semaphorin 3A-induced growth cone collapse.

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

Gene expression of neuropilin-1 and its receptors, VEGF/Semaphorin 3a, in normal and cancer cells.

Extracellular domains of the transmembrane glycoprotein, neuropilin-1 (Np1), specifically bind an array of factors and co-receptors including class-3 semaphorins (Sema3a), vascular endothelial growth factor (VEGF), hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-ß 1 (TGF-ß1), and fibroblast growth factor2 (FGF2). Np1 may have a role in immune response, tumor cell growth, and angiogenesis, but its relative expression in comparison to its co-primary receptors, VEGF and Sema3a, is not known. In this study we determined the mRNA expression of Np1 and its co-receptors, VEGF and Sema3a, and the ratio of VEGF/Sema3a in different human and rodent cell lines. Expression of Np1, VEGF and Sema3a is very low in cells derived from normal tissues, but these proteins are highly expressed in tumor-derived cells. Furthermore, the ratio of VEGF/Sema3a is highly variable in different tumor cells. The elevated mRNA expression of Np1 and its putative receptors in tumor cells suggests a role for these proteins in tumor cell migration and angiogenesis. As different tumor cells exhibit varying VEGF/Sema3a ratios, it appears that cancer cells show differential response to angiogenic factors. These results bring to light the individual variation among the cancer-related genes, Np1, VEGF, and Sema3a, and provide an important impetus for the possible personalized therapeutic approaches for cancer patients.

Effects of semaphorin 3A overexpression in corneal fibroblasts on the expression of adherens-junction proteins in corneal epithelial cells.

Interactions between epithelial cells and fibroblasts play important roles in tissue homeostasis. Semaphorins are a family of glycoproteins that contribute to axon guidance during development. With the use of a coculture system in which human corneal fibroblasts and epithelial cells are cultured on opposite sides of a collagen membrane, we have examined the effects of overexpression of semaphorin 3A in corneal fibroblasts on the expression of junctional proteins in corneal epithelial cells. Reverse transcription-polymerase chain reaction as well as immunoblot and immunofluorescence analyses revealed that the overexpression of semaphorin 3A in corneal fibroblasts increased the expression of E-cadherin and N-cadherin in corneal epithelial cells at both the mRNA and protein levels. These effects of semaphorin 3A were dependent on the presence of extracellular Ca(2+). Our findings indicate that semaphorin 3A released from corneal fibroblasts may play an important role in the regulation of intercellular communication between corneal epithelial cells as well as in the maintenance of corneal structure and function.

Expression of semaphorin 3A and its receptors in the human intervertebral disc: potential role in regulating neural ingrowth in the degenerate intervertebral disc.

INTRODUCTION: Intervertebral disc (IVD) degeneration is considered a major underlying factor in the pathogenesis of chronic low back pain. Although the healthy IVD is both avascular and aneural, during degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD, which may contribute to the pain. The mechanisms underlying neural ingrowth are, however, not fully understood. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves seeking their synaptic target. This study aimed to identify whether members of the Class 3 semaphorins were expressed by chondrocyte-like cells of the IVD addressing the hypothesis that they may play a role in repelling axons surrounding the healthy disc, thus maintaining its aneural condition. METHODS: Human IVD samples were investigated using reverse transcription polymerase chain reaction (RT-PCR) to identify gene expression of sema3A, 3F and their receptors: neuropilins (1 and 2) and plexins (A1-4). Sema3A protein was also localised within sections of normal and degenerate human IVD and immunopositivity quantified. Serial sections were stained using PGP9.5 and CD31 to correlate semaphorin 3A expression with nerve and blood vessel ingrowth, respectively. RESULTS: Sema3A protein was expressed highly in the healthy disc, primarily localised to the outer annulus fibrosus. In degenerate samples, sema3A expression decreased significantly in this region, although cell clusters within the degenerate nucleus pulposus exhibited strong immunopositivity. mRNA for sema3A receptors was also identified in healthy and degenerate tissues. CD31 and PGP9.5 were expressed most highly in degenerate tissues correlating with low expression of sema3A. CONCLUSIONS: This study is the first to establish the expression of semaphorins and their receptors in the human IVD with a decrease seen in the degenerate painful IVD. Sema3A may therefore, amongst other roles, act as a barrier to neuronal ingrowth within the healthy disc.

Psoralen-ultraviolet A therapy alters epidermal Sema3A and NGF levels and modulates epidermal innervation in atopic dermatitis.

BACKGROUND: Epidermal nerve densities are increased in patients with atopic dermatitis (AD), suggesting that it is partly responsible for the intense itching in the skin. Epidermal hyperinnervation in AD patients is decreased by ultraviolet (UV) phototherapy, although the underlying mechanisms are poorly understood. Interestingly, abnormal expression of axonal guidance molecules, such as nerve growth factor (NGF) and semaphorin 3A (Sema3A), is found in the epidermis of AD patients. Therefore, UV phototherapy may alter levels of axonal guidance molecule expression in atopic skin. OBJECTIVE: This study was performed to investigate whether epidermal Sema3A and NGF levels in AD are influenced by psoralen-UVA (PUVA) therapy. METHODS: Skin biopsies obtained from chronic AD patients before and after PUVA therapy were used. Both Sema3A and NGF in the skin were examined at mRNA and protein levels by quantitative RT-PCR and immunohistochemistry, respectively. Nerve fibers in the skin were stained with anti-PGP9.5 antibody, and the number of epidermal nerve fibers was counted. RESULTS: PUVA therapy decreased epidermal nerve densities in AD patients, concomitant with decreases in both visual analog scale (VAS) scores for pruritus and clinical severity scores. Increased fluorescence intensity of Sema3A and decreased fluorescence intensity of NGF were observed in the epidermis of PUVA-treated group. Moreover, Sema3A mRNA levels were upregulated in the PUVA-treated skins compared with untreated controls, while NGF mRNA levels in the skin were downregulated by the treatment. CONCLUSION: PUVA therapy may reduce epidermal hyperinnervation of AD by normalization of abnormal Sema3A and NGF expression in the epidermis.

Up-regulation of semaphorin 3A in human corneal fibroblasts by epidermal growth factor released from cocultured human corneal epithelial cells.

Semaphorins are a family of glycoproteins that play an important role in repulsive axon guidance during embryogenesis. We have now investigated the effect of corneal epithelial cells on the expression of Sema3A in corneal fibroblasts with the use of a coculture system in which the two cell types are separated by a collagen vitrigel membrane. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that the presence of immortalized human corneal epithelial (HCE) cells increased the expression of Sema3A in human corneal fibroblasts at both the mRNA and protein levels. This effect of HCE cells was mimicked by recombinant human epidermal growth factor (EGF) in a concentration- and time-dependent manner. An inhibitor of the tyrosine kinase activity of the EGF receptor, PD153035, blocked the EGF-induced up-regulation of both Sema3A mRNA and protein in corneal fibroblasts. Depletion of EGF in HCE cells by RNA interference largely abolished the effect of these cells on Sema3A expression in corneal fibroblasts. These findings indicate that EGF released from corneal epithelial cells up-regulates the expression of Sema3A in corneal fibroblasts. This effect of EGF may play an important role in maintenance of corneal structure and repair of corneal damage.

Different effects of mild and severe seizures on hippocampal neurogenesis in adult rats.

Recent evidence shows that functional neurogenesis exists in the adult hippocampus and that epileptic seizures can increase neurogenesis in the dentate gyrus (DG). However, it is unknown whether different seizure severity has different effects on neurogenesis in the DG of adult rats. In this study, we examined hippocampal neurogenesis in the rat mild and severe seizure preparations characterized with frequent wet dog shakes and severe status epilepticus, respectively. Both mild and severe seizures promoted the mitotic activity in the DG, but severe seizures caused a stronger cell proliferative response than mild seizures. Less than 20% of newborn cells in the DG differentiated into neurons in rats suffering severe seizures, whereas more than 60% of newborn dentate cells differentiated into neurons in control and mild seizure groups. Most newborn neurons migrated into the granular cell layer in control and mild seizure groups, but severe seizures were associated with an aberrant migration of newborn neurons into the dentate hilus. Severe seizures induced astrocyte activation and the expression of nestin and the migration directional molecules netrin 1 and Sema-3A in the hilus, which were not present in the hilus of control and mild seizure-attacked rats, suggesting that these molecules are involved in the aberrant migration of newborn neurons.